The therapeutic effect of miR-125b is enhanced by the prostaglandin endoperoxide synthase 2/cyclooxygenase 2 blockade and hampers ETS1 in the context of the microenvironment of bone metastasis

The therapeutic effect of miR-125b is enhanced by the prostaglandin endoperoxide synthase... Bone is the most common site for breast cancer spread. In the pro-metastatic cell line 1833, derived from MDA-MB-231 breast adenocarcinoma cells, both hypoxia and hepatocyte growth factor (HGF) influence the effect of miR-125b on ETS proto-oncogene 1 transcription factor (ETS1). The effect of hypoxia inducible factor 1 alpha subunit (HIF1A), known to promote metastatic spread by upregulating prostaglandin endoperoxide synthase 2 (PTGS2), may be dampened by miR-125b targeting PTGS2. Here, we investigated whether miR-125b plays a role in breast cancer metastasis by measuring its activity in response to the chemotherapeutic agent NS-398 in a xenograft model. NS-398 is typically used in the clinic to target PTGS2. We also aimed to describe the molecular mechanisms in vitro, since the enhancement of epithelial properties may favor the efficacy of therapies. We report that in the xenograft model, miR- 125b reduced metastasis to the bone. We also report suppression of PTGS2 enhanced survival by decreasing HIF1A in cells within the bone marrow. In 1833 cells transfected with a miR-125b mimic we observed several phenotypic changes including enhancement of the epithelial marker E-cadherin, a reduction of mesenchymal-associated genes and a reduction of WNT-associated stem cell signaling. Our findings suggest that in vivo, key players of the bone microenvironment promoting breast cancer spread are regulated by miR-125b. In future, biological molecules imitating miR-125b may enhance the sensitivity of chemotherapeutic agents used to counteract bone metastases. Introduction implicated through growth factors and various cellular Disseminated tumor cells (DTC) from breast carcinoma types of the staminal hematopoietic and osteoblastic 1,2 coordinate the formation of a favorable microenviron- niches . The presence of micrometastases, within the ment for future bone metastases, and the bone marrow is bone marrow premetastatic niche, is used as a predictive index for the survival of breast carcinoma patients; micrometastasis evolution leads to osteolytic metastases, involving the osteoclast-mediated reactivation of dormant Correspondence: Maria Alfonsina Desiderio (a.desiderio@unimi.it) 1 3,4 Istituto Ortopedico Galeazzi IRCCS, Via R. Galeazzi 4, 20161 Milano, Italy metastatic cells . Xenograft models are suitable to Dipartimento di Scienze Biomediche per la Salute, Molecular Pathology examine the molecular modifications in metastatic cells, Laboratory, Università degli Studi di Milano, Via L. Mangiagalli 31, 20133 Milano, supportive cells and extracellular matrix (ECM), which Italy Edited by G. Melino © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association 1234567890():,; 1234567890():,; Maroni et al. Cell Death and Disease (2018) 9:472 Page 2 of 13 underlie the interaction between metastasis and stroma. reducing also stem cell properties: this tool would permit Novel therapeutic approaches might counteract the to enhance the sensitivity to chemotherapeutic agents. favorable milieu of components in diseased bone after the Thus, the xenograft model of bone metastasis was pre- 5,6 DTC access . pared with 1833 cells transfected with miR-125b mimic, Microenvironmental stimuli contribute to the pheno- and the PTGS2 inhibitor N-(2-cyclohexyloxy-4-nitro- type plasticity, which is critical for the metastatic process phenyl)methanesulfonamide (NS-398) was administered 6–9 28 to the mice of DTC engrafted in the skeleton . In human bone . miR-125b mimic might hamper bone metastasis specimens, the hepatocyte growth factor metastasis colonization, by down-regulating ETS1 and (HGF)/Met receptor signaling pathway correlates with probably by affecting indirectly epithelial and stemness 10 11,13,29 miR-34a down-regulation . Since miRNAs play a role in genes , and might enhance the inhibitory effect of mRNA degradation or decrease of protein expression, and anti-PTGS2 chemotherapy. We studied E-cadherin, which this is consequence of the binding to the 3′-untranslated is a marker of epithelial phenotype of skeleton metastasis 11,12 18,19,30 region (3′UTR) (post-transcriptional level) , miR-34a from breast carcinoma , while the mesenchymal seems to target Met in skeleton metastases but not in the markers examined were matrix metalloproteinase2 pair-matched primary breast carcinomas . (MMP2), essential for dissemination of metastatic cells by The knowledge of miRNA functions in bone metastasis remodeling ECM (basal membrane and interstitial phenotype would be important for therapeutic pur- matrix), and vimentin which promotes adhesion/moti- 13,14 19,20 pose . miRNA deregulation seems to depend on the lity . The WNT pathway activity would indicate the microenvironment stimuli and/or on epigenetic control, acquirement of stem cell properties, and it is targeted by 18,29 and miRNA changes might inhibit or induce the epithelial miRNAs downstream of ETS1 . We expect that miR- 15–17 phenotype affecting invasion and metastasis . The 125b might interrupt the interaction between ETS1 and weakening of tumor cells adhesion, and the strengthening HIF1A , impairing directly and/or indirectly PTGS2, of cell motility and proteolysis trigger which is a target gene of HIF1 . ETS1 acts as a player for epithelial–mesenchymal transition (EMT) and invasion. the invasive program depending on the conditions of the 22,31 Notably, the mesenchymal phenotype and stemness are osseous microenvironment . 7,18–20 associated with neoplasia resistance to therapy . Under miR-125b and NS-398 combined treatment, we The miR-125b suppresses the osteogenic differentiation observed the reduction and delay of bone metastasis of mesenchymal stem cells derived from bone marrow , outgrowth, and decreases of the expression of ETS1, HIF1A and PTGS2 together with phenotypic changes. In and it targets specific signaling pathways such as those regulated by ETS proto-oncogene 1 transcription factor vitro, hypoxia and HGF interfered in the changes of gene (ETS1) . In 1833 bone metastatic cells, derived from expression due to miR-125b. MDA-MB-231 breast adenocarcinoma cells, the effects of miR-125b on ETS1 activity and on the biological func- Results tions are influenced by HGF and hypoxia, which are sti- Effect of miR-125b and NS-398 on bone metastasis growth muli of the bone microenvironment . miR-125b acting as Considering that miR-125b targets a wide spectrum of a tumor suppressor may translationally repress Human genes including PTGS2 and ETS1, and that miRNAs are antigen R (HuR), which affects the stability and transla- proposed as tools for cancer therapy, we tested miR-125b tional efficiency of target mRNAs . Thus, the expression alone or in combination with NS-398, a specific chemical of genes such as prostaglandin endoperoxide synthase 2 inhibitor of PTGS2 . In Figs. 1 and 2, we evaluated the (PTGS2) (previously known as cyclooxygenase 2), show- growth of bone metastasis and the survival of the xeno- ing three HuR binding sequences in the 3′UTR , might graft mice under these treatments. To prepare the xeno- be controlled by miR-125b. PTGS2 plays multiple roles in graft model, we used 1833-bone metastatic cells 23,24 primary tumors and bone metastases . In 1833 cells, engineered with the triple reporter construct with lumi- PTGS2 is implicated in the nuclear translocation of nescence (1833/TGL) . hypoxia inducible factor 1 alpha subunit (HIF1A) and in As shown in Fig. 1a, we performed the time-course of HIF1 activation and, therefore, in angiogenesis important bioluminescence images to evaluate the bone-metastasis for metastasis outgrowth . Also, miR-125b seems to development after 1833/TGL injection (ME): ME was the reverse multidrug resistance by modulating anti-apoptotic control mice group, and the effects of the single treat- factors . Even if some miRNA roles in cancer treatment ments or their combination were evaluated in respect to have been clarified , pre-clinical studies for antimeta- ME. Similar bioluminescence signals were observed for static therapies based on miRNA mimic are still lacking. ME and for the xenograft mice injected with miR-neg- The aim of the present paper is to provide the molecular CTR cells (data not shown). basis for the design of novel combined therapies to switch After quantification of the bioluminescence, the values metastatic cells towards the epithelial phenotype, for the hind limbs were reported in Fig. 1b. All the Official journal of the Cell Death Differentiation Association Maroni et al. Cell Death and Disease (2018) 9:472 Page 3 of 13 Fig. 1 miR-125b alone or in combination with NS-398 affected bone metastasis formation in the xenograft mice. a Representative bioluminescence imaging (BLI) of the xenograft mice treated as shown. ME, mice bearing bone metastases. b We report quantitative data for the bioluminescence signals, and the percent of inhibition due the treatments vs. ME. The data are the means ± S.E. of five mice per group. *P < 0.05, ***P < 0.001 vs. the bioluminescence value of ME at the corresponding time; °°P < 0.005 vs. the value under NS-398 treatment at the corresponding time; ◊ ◊◊ P < 0.05, P < 0.005 vs. the value under miR-125b at the corresponding time. c Representative images of the three-dimensional reconstruction of μCT at 21 and 33 days of treatment. Three xenograft mice per group were analyzed with similar results. The arrows indicate osteolytic lesions treatments did not practically affect metastatic growth bioluminescence signal at 26 days was reduced of about within 4 days from 1833/TGL injection. At 9 days, miR- 60% by the combined treatment (Supplementary 125b alone or in combination with NS-398 markedly Figure S1). prevented metastasis development, in respect to ME and Figure 2a shows that the exposure to miR-125b plus to NS-398 alone. Importantly, only for the combination NS-398 prolonged the mice survival of about 12 days in miR-125b plus NS-398 this strong inhibitory effect per- respect to that of ME, and the life-protective effect of the sisted until 21 days. therapy combination was evident also in comparison with In addition, we demonstrated by micro-computed the single treatments. tomography (μCT) analysis of the hind limbs that miR- Based on these data, we examined by immunohis- 125b plus NS-398 was effective in preventing at a great tochemistry at 26 days the expression of the target genes extent the osteolytic lesions at 21 days, making the of miR-125b as well as of HIF1A, known to be implicated comparison with ME at the same time (Fig. 1c). The single in PTGS2 regulation via HIF1 activity (Fig. 2b) .A treatments significantly reduced osteolysis at 21 days in semiquantitative evaluation of the immunohistochemistry the hind limbs. Of note, in the mice treated with miR- data was performed, considering low (2+), mild (3+), 125b plus NS-398 the osteolytic lesions were evidenced high (4+) and very high (5+) signals. The expression of again at 33 days (Fig. 1c). Consistently, the metastasis ETS1 and HIF1A was more elevated than that of PTGS2 wideness was less at 26 days than at 29 days, and the in ME-bone metastases: these gene signals were strongly Official journal of the Cell Death Differentiation Association Maroni et al. Cell Death and Disease (2018) 9:472 Page 4 of 13 combined treatment almost completely reduced the signal in the two compartments (Fig. 5). Negative controls did not show specific signals (Figs. 3–5). The effect of miR-125b or NS-398 treatment on ETS1, HIF1A and PTGS2 is reported in Supplementary Fig- ures S3-S5. In 1833 cells miR-125b mimic affected gene expression under the influence of hypoxia or HGF In vitro experiments were performed to better under- stand the molecular mechanisms underlying miR-125b effects in the bone metastasis xenograft model, and to clarify whether microenvironmental stimuli might play a key regulatory role. To this purpose, in 1833 cells trans- fected with miR-125b and exposed to hypoxia or HGF, we monitored epithelial, mesenchymal and stem cell mar- 7,18 kers , i.e., E-cadherin, MMP2, vimentin and WNT- pathway activity (Fig. 6). Also, we examined the expres- Fig. 2 Effect of miR-125b, NS-398 and their combination on sion of PTGS2, ETS1 and HIF1A, direct or indirect target xenograft mice survival, and on gene expression in bone genes of miR-125b, as well as of Secreted acidic and rich metastasis. a Survival curve on Kaplan-Meier plots of the data of treated and untreated ME. b Semi-quantitative evaluation of in cysteine (SPARC) and of osteocalcin, which seem 33,34 immunohistochemistry assays of ETS1, HIF1A and PTGS2 in bone involved in osteoblastic niche (Figs. 6 and 7) . Since we metastasis of xenograft mice treated as indicated. We analyzed serial have recently shown that ETS1 expression vector affects sections for each specimen from three mice per group of treatments HIF1A transactivation , miR-125b targeting ETS1 might at 26 days, and we found similar results influence HIF1A expression. miR-125b mimic augmented the E-cadherin expression only in the presence of 10% fetal bovine serum (FBS): this effect was counteracted by the concomitant exposure to hypoxia (Fig. 6a). Differently, HGF enhanced E-cadherin reduced (from 50 to 80%) by the combined exposure to miR-125b plus NS-398, while the single treatment miR- in 1833 cells transfected with miR-CTR, and miR-125b 125b or NS-398 seemed less effective. reduced E-cadherin under basal and HGF conditions. Altogether, our findings suggested a key role of the Hypoxia induced MMP2 and vimentin, while being genes examined in bone metastasis growth, and the effi- prevented by concomitant miR-125b transfection. Also, cacy of the combined treatment tested. miR-125b showed inhibitory effects on MMP2 and vimentin under 0.1% FBS, and HGF caused a reversion Expression of ETS1, HIF1A and PTGS2 and their (Fig. 6a). intracellular distribution in bone metastasis under miR- TOPFLASH-gene reporter was used to evaluate WNT 125b plus NS-398 pathway activity (Fig. 6b). miR-125b alone inhibited of Immunohistochemistry images of the genes examined about 40% the luciferase activity, while in the presence of are shown in Figs. 3–5. As reported in Fig. 3 and in hypoxia or HGF a reversion of TOPFLASH activity was Supplementary Figure S2, the expression of ETS1 was observed, peaking (2.5-fold) under hypoxia. very high in bone-metastasis both at cytosol and nuclear In the nuclei of the cells transfected with miR-CTR, the levels (inset). ETS1 was elevated also in the cytosol of long-time exposure to hypoxia gave a huge HIF1A supportive cells of the bone marrow, while in the normal increase, and HGF tripled HIF1A: these increases were bone the ETS1 signal was scarce (Supplementary Fig- partly prevented by the concomitant miR-125b treatment. ure S2). The miR-125b plus NS-398 treatment reduced The transfection of miR-125b under 10% FBS (upper mostly metastatic ETS1, including the nuclear signal panel) and 0.1% FBS (lower panel) doubled HIF1A in the (Fig. 3, insets). As shown in Fig. 4, in bone metastases cytosol (Fig. 6c). under the combined treatment, the expression of HIF1A Altogether, the 1833 cells required the serum presence largely diminished at cytosol and nuclear levels (insets); for the miR-125b-dependent stimulation of the epithelial the signal of HIF1A extensively decreased also in the marker E-cadherin, while the miR-125b negative effects cytosol of the bone marrow supportive cells. PTGS2 signal towards the mesenchymal markers MMP2 and vimentin, in bone metastases was mild, showing prevalent cytosolic the staminal WNT pathway and nuclear HIF1A occurred localization, and it was low in the bone marrow: the both under 10 and 0.1% FBS. Also, the reduction of Official journal of the Cell Death Differentiation Association Maroni et al. Cell Death and Disease (2018) 9:472 Page 5 of 13 Fig. 3 miR-125b in combination with NS-398 affected ETS1 expression in bone metastasis of xenograft mice. We show representative images of ETS1 in bone metastasis from mice treated with the combination miR-125b plus NS-398, or not (ME). Serial sections for each specimen from three mice at 26 days were analyzed obtaining similar results. me, metastasis; bm, bone marrow; bo, bone. Scale bar = 120 μm (reported in 40× magnification panels). Scale bar = 60 μm (reported in the three insets). The negative control was performed without the specific antibody Official journal of the Cell Death Differentiation Association Maroni et al. Cell Death and Disease (2018) 9:472 Page 6 of 13 Fig. 4 HIF1A expression in bone metastasis of xenograft mice under miR-125b in combination with NS-398. We show representative images of HIF1A in bone metastasis of mice treated with the combination miR-125b plus NS-398, or not (ME). Serial sections for each specimen from three mice at 26 days were analyzed obtaining similar results. me, metastasis; bm, bone marrow; bo, bone. Scale bar = 120 μm (reported in an exemplificative panel). The negative control was performed without the specific antibody nuclear HIF1A by miR-125b in the presence of hypoxia or Figure 7a shows that 36-h hypoxia doubled the ETS1 HGF, was consistent with the immunohistochemistry protein level in 1833 cells, but not when miR-CTR was findings (Supplementary Figure S4). transfected. miR-125b in the presence or the absence of Official journal of the Cell Death Differentiation Association Maroni et al. Cell Death and Disease (2018) 9:472 Page 7 of 13 Fig. 5 miR-125b in combination with NS-398 affected PTGS2 expression in bone metastasis of xenograft mice. We show representative images of PTGS2 in bone metastasis of mice treated with the combination miR-125b plus NS-398, or not (ME). Serial sections for each specimen from three mice at 26 days were analyzed obtaining similar results. me, metastasis; bm, bone marrow; bo, bone. Scale bar = 120 μm (reported in an exemplificative panel). The negative control was performed without the specific antibody hypoxia down-regulated ETS1 (p51/52), while the p27 (Fig. 7b). The consensus sequences for transcription factors form of ETS1 was unchanged by the treatments tested. in osteocalcin and SPARC promoters are reported in Sup- PTGS2, SPARC and osteocalcin were induced by hypoxia plementary Figure S6: the numerous ETS1 and Runx2 in 1833 cells transfected or not with miR-CTR: miR-125b binding sites shown might explain the efficacy of the largely prevented the stimulatory effects of hypoxia treatments affecting metastasis phenotype. Putative binding Official journal of the Cell Death Differentiation Association Maroni et al. Cell Death and Disease (2018) 9:472 Page 8 of 13 Fig. 6 Effect of miR-125b on epithelial and mesenchymal markers and on HIF1A expression under hypoxia or HGF in 1833 cells. a Histograms of Western blots densitometric data; the experiments were repeated three times and the means ± S.E. are shown. Vinculin was used for Δ ΔΔ normalization. P < 0.05, P < 0.005 vs. miR-CTR; *P < 0.05 vs. miR-125b. Also, we report the representative images of the Western blots. b TOPFLASH luciferase activity is shown. The assays were performed in triplicate, and were repeated three times; the data are shown as the means ± S.E. P < 0.05 vs. miR-CTR; *P < 0.05 vs. miR-125b. c Representative images of Western blots performed in triplicate are shown. Vinculin and B23 were used for Δ ΔΔ normalization. The data of densitometric analysis are reported in the table as fold-variations vs. the first lane, considered 1. P < 0.05, P < 0.005 vs. § §§ miR-CTR; *P < 0.05 vs. miR-CTR plus hypoxia or plus HGF; P < 0.05, P < 0.005 vs. miR-125b sites of Twist and Snail were also present in the two gene of ETS1. In 1833 cells, HGF induced the dominant promoters, suggesting a relationship between the changes of negative p27, which might interfere in ETS1 activity gene expression and the metastatic phenotype. (Fig. 7c). miR-125b transfection, in the presence or the absence of The pattern of ETS1 under HGF or hypoxia in vitro was HGF, down-regulated both the p51/52 and the p27 forms in agreement with that observed in vivo, indicating the Official journal of the Cell Death Differentiation Association Maroni et al. Cell Death and Disease (2018) 9:472 Page 9 of 13 Fig. 7 A gene pattern for adhesion/invasion of 1833-bone metastatic cells was affected by miR-125b under hypoxia, or HGF treatment. a Representative images of Western blots repeated three times are shown. Vinculin was used for normalization. The data of densitometric analysis are Δ § reported in the table as fold-variations vs. the first lane, considered 1. *P < 0.05 vs. untreated 1833 cells; P < 0.05 vs. miR-CTR; P < 0.05 vs. miR-CTR under hypoxia. b Representative images of Western blots repeated three times are shown. Vinculin was used for normalization. The numbers at the Δ ΔΔ § bottom indicate the fold-variations vs. the first lane, considered 1. *P < 0.05, **P < 0.005 vs. untreated 1833 cells; P < 0.05, P < 0.005 vs. miR-CTR; P < 0.05 vs. miR-CTR under hypoxia. c Representative images of Western blots repeated three times are shown. Vinculin was used for normalization. The data of densitometric analysis are reported in the table as fold-variations vs. the first lane, considered 1. *P < 0.05 vs. untreated 1833 cells; P < 0.05 vs. miR-CTR; P < 0.05 vs. miR-CTR under HGF. d Representative images of Western blots repeated three times are shown. Vinculin was used for Δ ΔΔ normalization. The numbers at the bottom indicate the fold-variations vs. the first lane, considered 1. *P < 0.05 vs. untreated 1833 cells; P < 0.05, P < 0.005 vs. miR-CTR; P < 0.05 vs. miR-CTR under HGF. e To make a comparative evaluation of the gene effects of miR-125b under hypoxia or HGF, we show the histograms of the densitometric values of the Western blots for PTGS2, SPARC and osteocalcin. The data are the means ± S.E. of three Δ ΔΔ § separate experiments. P < 0.05, P < 0.005 vs. respective miR-CTR transfected cells; P < 0.05 vs. respective miR-125b transfected cells Official journal of the Cell Death Differentiation Association Maroni et al. Cell Death and Disease (2018) 9:472 Page 10 of 13 important role played by these microenvironmental sti- muli in the inhibitory effect of miR-125b towards ETS1 in the xenograft model (Supplementary Figure S3). Only under HGF but not in hypoxic conditions the ETS1-p27 form decreased when the 1833 cells were transfected with miR-CTR or miR-125b. PTGS2 and SPARC were induced by miR-125b plus HGF; osteocalcin was strongly down-regulated by star- vation, and incremented under miR-125b plus HGF (Fig. 7d). In conclusion, in the histograms (Fig. 7e) we compared the effects of miR-125b under the two microenviron- Fig. 8 Effects of miR-125b on gene expression under mental stimuli on PTGS2, SPARC and osteocalcin. microenvironmental stimuli in bone metastasis SPARC is a matricellular protein, not really an ECM component, which might participate in the stiffness and the release of growth factors , initiating changes in neoplastic cell phenotype with an involvement of miR- at early stages after bone engraftment of DTC. The NAs . Osteocalcin is important for the osteoblastic therapeutic approach consisting in the overexpression of mineralization of endosteal-like ECM: the endosteum miR-125b which targets a wide spectrum of genes, such as lines the inner wall of long bones surrounding the bone ETS1 and PTGS2, will eventually impact clinical practice marrow cavity, and drives the cellular changes for osteo- preventing bone resorption. The exit from quiescence/ 4,33 lytic metastases . Normally, osteocalcin is produced dormancy depends on osteoclast activation, and also on exclusively by osteoblasts, acting in the bone matrix as a stroma composition and neoangiogenic vessels . non-collagenous protein, and is now considered an inhi- The molecular mechanisms underlying the therapeutic bitor of bone mineralization even if opposite hypotheses efficacy of the studied treatment miR-125b plus NS-398 cannot be excluded . By considering PTGS2 and SPARC, would be multiple (Fig. 8). First, the blockade of the miR-125b was stimulatory in the presence of HGF and autoregulatory loop between ETS1 and HIF1A/HIF1, inhibitory under hypoxia. Osteocalcin seemed strongly because of the reciprocal control of these transcription hampered by miR-125b, mostly under starvation, with a factor activities , and second a complete inhibition of partial reversion both under HGF and hypoxia. Thus, the PTGS2 by the concomitant chemical and miR-125b effect of miR-125b on target genes seemed to depend on treatment. At support, PTGS2 signal remarkably microenvironmental stimuli. decreased in osseous metastases and the bone marrow, also due to the down-regulation of HIF1A throughout the Discussion metastatic tissue: the alpha/beta heterodimer HIF1 is a In the 1833 xenograft model of skeleton metastasis from transcription factor known to target PTGS2 and 20,25 breast carcinoma the concomitant exposure to miR-125b ETS1 . Our proposed therapy would have a direct mimic and NS-398, a specific PTGS2 chemical inhibitor, effect on PTGS2, through miR-125b action on HuR sites, reduced and delayed the outgrowth of osteolytic bone even if a post-transcriptional effect of miR-125b on ETS1 metastases prolonging mice survival in respect to the cannot be excluded. Also, an indirect effect of the therapy single treatments. The miR-125b strongly enhanced the on ETS1 through HIF1A downregulation might occur, life-protective effect of NS-398, and we hypothesize that consistent with the decreases of ETS1 and HIF1A in the efficacy of the combined treatment depended on the cytosol and nuclei of metastases and in the bone marrow. reduction of chemoresistance, known to be associated We suggest that these amplifying effects might impinge 7,18 with EMT . The endpoint of our study was to on invasion and angiogenesis, because ETS1 and PTGS2 demonstrate that miR-125b enhances the epithelial-like are key players in these processes: PTGS2 through pros- transition in vitro, and that the paracrine stimuli and taglandin E2 regulates HIF1A nuclear translocation and 20,25 physical conditions might counteract or favor the phe- HIF1 activity (Supplementary Figure S4) . Capillaries notypic changes, explaining also the in vivo findings on are associated with quiescent and actively resorbing growth and gene expression in bone metastases. endosteal surfaces, and are implicated in development of Importantly, the adverse effect of miR-125b mimic plus bone metastasis, permitting the access of DTC to bone- NS-398 on metastatic growth started at 4 days from 1833/ remodeling compartment or to the immediate adjacent TGL-cell injection and became highly significant at microenvironment . 9 days, leading to suppose that the hampering of coloni- Third, in vivo phenotypic changes under miR-125b zation was caused by molecular and cellular modifications plus NS-398 seemed to depend on the metastatic Official journal of the Cell Death Differentiation Association Maroni et al. Cell Death and Disease (2018) 9:472 Page 11 of 13 down-regulations of ETS1 and HIF1A, which are critical the stimulatory effect of hypoxia. Therefore, the miR-125b 20,39 for the invasive phenotype and staminality . Also, in mimic might reduce bone metastasis colonization in vivo agreement with the hypothesis that in 1833-xenograft influencing SPARC roles in metastasis plasticity and in model miR-125b decreased the metastasis resistance to metastasis dormancy, by the regulation of biological and 34,36,46,47 chemotherapy, the microenvironmental stimuli would be physical properties of the ECM . HIF1A beyond critical for the effect of miR-125b in vivo, suggesting that SPARC participates in metastasis dormancy . the bone context encountered by DTC would be funda- In conclusion, a strong down-regulation of osteocalcin mental for their fate. The skeleton microenvironment is was observed in the presence of miR-125b under serum modified by the engraftment of metastatic cells influen- deprivation, indicating the susceptibility to growth factors, 18,37 cing the EMT , and it would interfere in the therapies while HGF restoration not only reversed osteocalcin such as that with miR-125b mimic. expression, but also increased PTGS2 and SPARC under The axis HGF/Met receptor seems to be implicated in miR-125b. The miR-125b-dependent impairment of 20 38 stroma-induced drug desensitization , and hypoxia con- osteocalcin, considered an inhibitor of mineralization , ferring stem like properties might give resistance to might be related to protection from osteolysis: this effect therapy . Consistently, we found that the tumor sup- would be partly prevented by the microenvironmental pressor gene miR-125b in 1833 cells reduced WNT stimuli. The combined therapeutic approach miR-125b activity and targeted transcription factors, such as ETS1, plus NS-398 seems promising for the early hampering HIF1 and WNT, likely affecting gene expression in bone effects on metastatic growth and osteolytic lesions, con- metastasis. Numerous ETS1 consensus sequences are sidering also that the initial metastatic steps and therapy present in SPARC and osteocalcin promoters (Supple- efficacy are strongly influenced by the microenvironment mentary Figure S6). In addition, considering that in bone- conditions. metastatic cells miR-125b enhanced the expression of E- cadherin, while decreasing MMP2 and vimentin, our Materials and Methods present results suggested a switch towards an epithelial Cell transfection phenotype, which is known to be more responsive to The 1833 bone metastatic clone, derived from MDA- chemotherapy than the mesenchymal/invasive MB-231 breast adenocarcinoma cells, and the 1833 cells 7,18,20 phenotype . retrovirally transfected with HSV1-tk/GFP/firefly lucifer- In bone metastatic cells, which show an epithelial phe- ase construct (1833/TGL) , were a gift from Dr. J. Mas- notype , miR-125b might antagonize the function of sagué (Memorial Sloan-Kettering Cancer Center, New miR-200 : we propose that miR-125b by blocking p53 York, NY, USA). 11,19,37 prevents miR-200-inhibitory function . As a con- The cells, routinely maintained in DMEM containing sequence, Twist activity might mediate the MET meta- 10% FBS (Sigma-Aldrich, Saint Louis, MO), were used stable phenotype with E-cadherin expression . The miR- after two or three passages in culture. The 1833 and 1833/ 125b effect on the gene expression of bone metastatic TGL cells were infected with miR-125b expression lenti- cells depended on serum conditions, HGF and hypoxia virus (hsa-miR-125b-5p MIMAT0000423, GenTarget exposure, favoring the reduction of ETS1 expression Inc., San Diego, CA, USA) or with miR-CTR (negative in vitro, while the microenvironmental stimuli counter- control, empty miR lentivirus, GenTarget Inc.), following acted the miR-125b-dependent epithelial phenotype with the manufacturer’s recommended procedures. Lentiviral E-cadherin reduction by hypoxia and MMP2 and vectors co-expressed puromycin resistance for the selec- vimentin enhancements by HGF. tion of infected cells. A pilot experiment has been done to Altogether, the knowledge of phenotypic changes under determine the antibiotic’s kill curve for our cells. Thus, therapy were further deepened by studying SPARC and 72-h infected cells were selected with 2 μg/ml puromycin, osteocalcin, which are target genes of Runx2 transcription and were maintained under selection in all the 34,41 factor ; HIF1A expression is also controlled by Runx2 experiments. by preventing the ubiquitination . Runx2 plays key roles In another group of experiments, flasks containing 1833 43,44 in bone metastasis from breast carcinoma , being cells at 70% of confluence were used to transfect the 35,45 regulated by Endothelin-1, HGF and hypoxia . Nota- TOPFLASH gene reporter from Dr. B.M. Gumbiner bly, bone-metastatic cells acquire osteomimicry by pro- (Memorial Sloan-Kettering Cancer Center, New York, ducing SPARC and osteocalcin, as shown in the 1833- NY, USA). For the transfection of the TOPFLASH con- 32,34,46 xenograft model . struct, we performed an incubation mixture 3:1, DNA: HGF and hypoxia caused SPARC accumulation, which Fugene 6 (Roche Diagnostics, Monza, Italy); the mixture might be important to determine the adhesive properties contained also the internal control pRL-TK (Renilla of bone metastasis in the osteoblastic niche, ECM stiffness Luciferase Plasmid). Firefly/Renilla luciferase activity and growth-factor release, and miR-125b counteracted ratios were calculated by the software . Official journal of the Cell Death Differentiation Association Maroni et al. Cell Death and Disease (2018) 9:472 Page 12 of 13 Animal treatment Transduction Laboratories, Bedford, MA, USA), anti- Animal studies were carried out according to the MMP2 (1:500, Novus Biologicals, Abingdon, UK), anti- Institutional Guide for Care and Use of Laboratory vimentin (1:500 Santa-Cruz Biotechnology), anti-SPARC Animals, and the Directive 2010/63/UE. All mice were (1:200, H-90 Santa Cruz Biotechnology), anti-osteocalcin bred and maintained under specific pathogen-free con- (5 μg/ml, Abcam, Cambridge, UK), anti-B23 (1:1000, ditions in the institutional animal facility of the Scientific H106 Santa Cruz Biotechnology), or anti-vinculin (1:1000, Cell Signaling, Leiden, The Netherlands) was used for Institute San Raffaele, Milano, Italy. All experiments were performed with the protocol approved by the Institutional immunoblotting. Densitometric analysis was performed Animal Care and Use Committee of Scientific Institute after reaction with ECL plus chemiluminescence kit from San Raffaele and by Ministero della Salute. Anaesthetized Thermo-Fisher Scientific (Waltham, MA, USA) or Clarity nu/nu mice were treated as follows: one animal group Max Western ECL Substrate-Luminol Solution (Biorad, (ME, n = 11) was injected with the 1833/TGL cells; a Hercules, CA, USA). second group (ME + miR-125b, n = 11) received 1833/ TGL cells infected with miR-125b; a third group (ME + Statistical analysis miR-125b + NS-398, n = 10) received 1833/TGL cells The statistical analysis of the values of bioluminescence infected with miR-125b, and the mice were concomitantly for the xenograft mice groups, as well as of Western injected with 20 mg/kg NS-398 i.p. 5 days/week until the blots densitometry and luciferase activity values was suppression; the fourth group was treated only with performed by analysis of variance. The number of NS-398 (n = 10); the fifth group was administered independent experiments has been indicated in the 1833/TGL cells infected miR-neg-CTR (ME + miR-CTR, Legends of the Figures. The data are shown as means ± n = 4). For the xenograft model preparation, 5 × 10 S.E., and their significance was evaluated on the infected or non-infected cells were injected to the mice, original values in the case of fold-variations. P < 0.05 was that were monitored by Optical imaging and μCT assay , considered significant. For xenograft mice, the survival and were sacrificed to prevent the suffering. Three ani- data were analyzed by Kaplan–Meier method and the mals per group were sacrificed at the indicated times to Log-rank (Mantel–Cox) test. P < 0.05 was considered perform immunohistochemistry and Hematoxylin & significant. Eosin assays. Acknowledgements The injection efficiency for all the mice was controlled This work was supported by grants from Ministero della Salute Ricerca by monitoring the bioluminescence signal 1 h after Corrente L4077, L4084 and L4101. xenografting. We normalized the data of bioluminescence Author details for each animal with the value obtained at 24 h. This Istituto Ortopedico Galeazzi IRCCS, Via R. Galeazzi 4, 20161 Milano, Italy. procedure was made to exclude a potential impact of the 2 Dipartimento di Scienze Biomediche per la Salute, Molecular Pathology pretreatments, i.e., miR-125b-cell transfection or NS-398 Laboratory, Università degli Studi di Milano, Via L. Mangiagalli 31, 20133 Milano, Italy exposure, on extravasation and homing with an inter- ference in the evaluation of metastasis growth. Conflict of Interest The authors declare that they have no conflict of interest. Immunohistochemistry Bone samples fixed with 10% formalin, were decalcified, Publisher's note embedded in paraffin, and serial sections were prepared . Springer Nature remains neutral with regard to jurisdictional claims in To evaluate ETS1, HIF1A and PTGS2, the immunos- published maps and institutional affiliations. taining was performed with anti-Ets-1 (1:50, C20 Santa Supplementary Information accompanies this paper at https://doi.org/ Cruz Biotechnology, Santa Cruz, CA, USA), anti-HIF-1α 10.1038/s41419-018-0499-8. (1:100, Novus Biologicals, Littleton, CO, USA), and anti- COX2 (1:50, Cayman Chemical, Ann Arbor, MI, USA). Received: 30 November 2017 Revised: 14 March 2018 Accepted: 16 March Western blot assay Some flasks containing 1833 cells infected with miR- 125b mimic were exposed to hypoxia , or to 100 ng/ml References HGF under starvation , and total and nuclear protein 1. Weilbaecher, K. N., Guise, T. A. & McCauley, L. K. Cancer to bone: a fatal extracts were prepared. To evaluate ETS1, HIF1A and attraction. Nat. Rev. Cancer 11,411–425 (2011). 2. Dhawan,A.et al. 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The therapeutic effect of miR-125b is enhanced by the prostaglandin endoperoxide synthase 2/cyclooxygenase 2 blockade and hampers ETS1 in the context of the microenvironment of bone metastasis

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Abstract

Bone is the most common site for breast cancer spread. In the pro-metastatic cell line 1833, derived from MDA-MB-231 breast adenocarcinoma cells, both hypoxia and hepatocyte growth factor (HGF) influence the effect of miR-125b on ETS proto-oncogene 1 transcription factor (ETS1). The effect of hypoxia inducible factor 1 alpha subunit (HIF1A), known to promote metastatic spread by upregulating prostaglandin endoperoxide synthase 2 (PTGS2), may be dampened by miR-125b targeting PTGS2. Here, we investigated whether miR-125b plays a role in breast cancer metastasis by measuring its activity in response to the chemotherapeutic agent NS-398 in a xenograft model. NS-398 is typically used in the clinic to target PTGS2. We also aimed to describe the molecular mechanisms in vitro, since the enhancement of epithelial properties may favor the efficacy of therapies. We report that in the xenograft model, miR- 125b reduced metastasis to the bone. We also report suppression of PTGS2 enhanced survival by decreasing HIF1A in cells within the bone marrow. In 1833 cells transfected with a miR-125b mimic we observed several phenotypic changes including enhancement of the epithelial marker E-cadherin, a reduction of mesenchymal-associated genes and a reduction of WNT-associated stem cell signaling. Our findings suggest that in vivo, key players of the bone microenvironment promoting breast cancer spread are regulated by miR-125b. In future, biological molecules imitating miR-125b may enhance the sensitivity of chemotherapeutic agents used to counteract bone metastases. Introduction implicated through growth factors and various cellular Disseminated tumor cells (DTC) from breast carcinoma types of the staminal hematopoietic and osteoblastic 1,2 coordinate the formation of a favorable microenviron- niches . The presence of micrometastases, within the ment for future bone metastases, and the bone marrow is bone marrow premetastatic niche, is used as a predictive index for the survival of breast carcinoma patients; micrometastasis evolution leads to osteolytic metastases, involving the osteoclast-mediated reactivation of dormant Correspondence: Maria Alfonsina Desiderio (a.desiderio@unimi.it) 1 3,4 Istituto Ortopedico Galeazzi IRCCS, Via R. Galeazzi 4, 20161 Milano, Italy metastatic cells . Xenograft models are suitable to Dipartimento di Scienze Biomediche per la Salute, Molecular Pathology examine the molecular modifications in metastatic cells, Laboratory, Università degli Studi di Milano, Via L. Mangiagalli 31, 20133 Milano, supportive cells and extracellular matrix (ECM), which Italy Edited by G. Melino © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association 1234567890():,; 1234567890():,; Maroni et al. Cell Death and Disease (2018) 9:472 Page 2 of 13 underlie the interaction between metastasis and stroma. reducing also stem cell properties: this tool would permit Novel therapeutic approaches might counteract the to enhance the sensitivity to chemotherapeutic agents. favorable milieu of components in diseased bone after the Thus, the xenograft model of bone metastasis was pre- 5,6 DTC access . pared with 1833 cells transfected with miR-125b mimic, Microenvironmental stimuli contribute to the pheno- and the PTGS2 inhibitor N-(2-cyclohexyloxy-4-nitro- type plasticity, which is critical for the metastatic process phenyl)methanesulfonamide (NS-398) was administered 6–9 28 to the mice of DTC engrafted in the skeleton . In human bone . miR-125b mimic might hamper bone metastasis specimens, the hepatocyte growth factor metastasis colonization, by down-regulating ETS1 and (HGF)/Met receptor signaling pathway correlates with probably by affecting indirectly epithelial and stemness 10 11,13,29 miR-34a down-regulation . Since miRNAs play a role in genes , and might enhance the inhibitory effect of mRNA degradation or decrease of protein expression, and anti-PTGS2 chemotherapy. We studied E-cadherin, which this is consequence of the binding to the 3′-untranslated is a marker of epithelial phenotype of skeleton metastasis 11,12 18,19,30 region (3′UTR) (post-transcriptional level) , miR-34a from breast carcinoma , while the mesenchymal seems to target Met in skeleton metastases but not in the markers examined were matrix metalloproteinase2 pair-matched primary breast carcinomas . (MMP2), essential for dissemination of metastatic cells by The knowledge of miRNA functions in bone metastasis remodeling ECM (basal membrane and interstitial phenotype would be important for therapeutic pur- matrix), and vimentin which promotes adhesion/moti- 13,14 19,20 pose . miRNA deregulation seems to depend on the lity . The WNT pathway activity would indicate the microenvironment stimuli and/or on epigenetic control, acquirement of stem cell properties, and it is targeted by 18,29 and miRNA changes might inhibit or induce the epithelial miRNAs downstream of ETS1 . We expect that miR- 15–17 phenotype affecting invasion and metastasis . The 125b might interrupt the interaction between ETS1 and weakening of tumor cells adhesion, and the strengthening HIF1A , impairing directly and/or indirectly PTGS2, of cell motility and proteolysis trigger which is a target gene of HIF1 . ETS1 acts as a player for epithelial–mesenchymal transition (EMT) and invasion. the invasive program depending on the conditions of the 22,31 Notably, the mesenchymal phenotype and stemness are osseous microenvironment . 7,18–20 associated with neoplasia resistance to therapy . Under miR-125b and NS-398 combined treatment, we The miR-125b suppresses the osteogenic differentiation observed the reduction and delay of bone metastasis of mesenchymal stem cells derived from bone marrow , outgrowth, and decreases of the expression of ETS1, HIF1A and PTGS2 together with phenotypic changes. In and it targets specific signaling pathways such as those regulated by ETS proto-oncogene 1 transcription factor vitro, hypoxia and HGF interfered in the changes of gene (ETS1) . In 1833 bone metastatic cells, derived from expression due to miR-125b. MDA-MB-231 breast adenocarcinoma cells, the effects of miR-125b on ETS1 activity and on the biological func- Results tions are influenced by HGF and hypoxia, which are sti- Effect of miR-125b and NS-398 on bone metastasis growth muli of the bone microenvironment . miR-125b acting as Considering that miR-125b targets a wide spectrum of a tumor suppressor may translationally repress Human genes including PTGS2 and ETS1, and that miRNAs are antigen R (HuR), which affects the stability and transla- proposed as tools for cancer therapy, we tested miR-125b tional efficiency of target mRNAs . Thus, the expression alone or in combination with NS-398, a specific chemical of genes such as prostaglandin endoperoxide synthase 2 inhibitor of PTGS2 . In Figs. 1 and 2, we evaluated the (PTGS2) (previously known as cyclooxygenase 2), show- growth of bone metastasis and the survival of the xeno- ing three HuR binding sequences in the 3′UTR , might graft mice under these treatments. To prepare the xeno- be controlled by miR-125b. PTGS2 plays multiple roles in graft model, we used 1833-bone metastatic cells 23,24 primary tumors and bone metastases . In 1833 cells, engineered with the triple reporter construct with lumi- PTGS2 is implicated in the nuclear translocation of nescence (1833/TGL) . hypoxia inducible factor 1 alpha subunit (HIF1A) and in As shown in Fig. 1a, we performed the time-course of HIF1 activation and, therefore, in angiogenesis important bioluminescence images to evaluate the bone-metastasis for metastasis outgrowth . Also, miR-125b seems to development after 1833/TGL injection (ME): ME was the reverse multidrug resistance by modulating anti-apoptotic control mice group, and the effects of the single treat- factors . Even if some miRNA roles in cancer treatment ments or their combination were evaluated in respect to have been clarified , pre-clinical studies for antimeta- ME. Similar bioluminescence signals were observed for static therapies based on miRNA mimic are still lacking. ME and for the xenograft mice injected with miR-neg- The aim of the present paper is to provide the molecular CTR cells (data not shown). basis for the design of novel combined therapies to switch After quantification of the bioluminescence, the values metastatic cells towards the epithelial phenotype, for the hind limbs were reported in Fig. 1b. All the Official journal of the Cell Death Differentiation Association Maroni et al. Cell Death and Disease (2018) 9:472 Page 3 of 13 Fig. 1 miR-125b alone or in combination with NS-398 affected bone metastasis formation in the xenograft mice. a Representative bioluminescence imaging (BLI) of the xenograft mice treated as shown. ME, mice bearing bone metastases. b We report quantitative data for the bioluminescence signals, and the percent of inhibition due the treatments vs. ME. The data are the means ± S.E. of five mice per group. *P < 0.05, ***P < 0.001 vs. the bioluminescence value of ME at the corresponding time; °°P < 0.005 vs. the value under NS-398 treatment at the corresponding time; ◊ ◊◊ P < 0.05, P < 0.005 vs. the value under miR-125b at the corresponding time. c Representative images of the three-dimensional reconstruction of μCT at 21 and 33 days of treatment. Three xenograft mice per group were analyzed with similar results. The arrows indicate osteolytic lesions treatments did not practically affect metastatic growth bioluminescence signal at 26 days was reduced of about within 4 days from 1833/TGL injection. At 9 days, miR- 60% by the combined treatment (Supplementary 125b alone or in combination with NS-398 markedly Figure S1). prevented metastasis development, in respect to ME and Figure 2a shows that the exposure to miR-125b plus to NS-398 alone. Importantly, only for the combination NS-398 prolonged the mice survival of about 12 days in miR-125b plus NS-398 this strong inhibitory effect per- respect to that of ME, and the life-protective effect of the sisted until 21 days. therapy combination was evident also in comparison with In addition, we demonstrated by micro-computed the single treatments. tomography (μCT) analysis of the hind limbs that miR- Based on these data, we examined by immunohis- 125b plus NS-398 was effective in preventing at a great tochemistry at 26 days the expression of the target genes extent the osteolytic lesions at 21 days, making the of miR-125b as well as of HIF1A, known to be implicated comparison with ME at the same time (Fig. 1c). The single in PTGS2 regulation via HIF1 activity (Fig. 2b) .A treatments significantly reduced osteolysis at 21 days in semiquantitative evaluation of the immunohistochemistry the hind limbs. Of note, in the mice treated with miR- data was performed, considering low (2+), mild (3+), 125b plus NS-398 the osteolytic lesions were evidenced high (4+) and very high (5+) signals. The expression of again at 33 days (Fig. 1c). Consistently, the metastasis ETS1 and HIF1A was more elevated than that of PTGS2 wideness was less at 26 days than at 29 days, and the in ME-bone metastases: these gene signals were strongly Official journal of the Cell Death Differentiation Association Maroni et al. Cell Death and Disease (2018) 9:472 Page 4 of 13 combined treatment almost completely reduced the signal in the two compartments (Fig. 5). Negative controls did not show specific signals (Figs. 3–5). The effect of miR-125b or NS-398 treatment on ETS1, HIF1A and PTGS2 is reported in Supplementary Fig- ures S3-S5. In 1833 cells miR-125b mimic affected gene expression under the influence of hypoxia or HGF In vitro experiments were performed to better under- stand the molecular mechanisms underlying miR-125b effects in the bone metastasis xenograft model, and to clarify whether microenvironmental stimuli might play a key regulatory role. To this purpose, in 1833 cells trans- fected with miR-125b and exposed to hypoxia or HGF, we monitored epithelial, mesenchymal and stem cell mar- 7,18 kers , i.e., E-cadherin, MMP2, vimentin and WNT- pathway activity (Fig. 6). Also, we examined the expres- Fig. 2 Effect of miR-125b, NS-398 and their combination on sion of PTGS2, ETS1 and HIF1A, direct or indirect target xenograft mice survival, and on gene expression in bone genes of miR-125b, as well as of Secreted acidic and rich metastasis. a Survival curve on Kaplan-Meier plots of the data of treated and untreated ME. b Semi-quantitative evaluation of in cysteine (SPARC) and of osteocalcin, which seem 33,34 immunohistochemistry assays of ETS1, HIF1A and PTGS2 in bone involved in osteoblastic niche (Figs. 6 and 7) . Since we metastasis of xenograft mice treated as indicated. We analyzed serial have recently shown that ETS1 expression vector affects sections for each specimen from three mice per group of treatments HIF1A transactivation , miR-125b targeting ETS1 might at 26 days, and we found similar results influence HIF1A expression. miR-125b mimic augmented the E-cadherin expression only in the presence of 10% fetal bovine serum (FBS): this effect was counteracted by the concomitant exposure to hypoxia (Fig. 6a). Differently, HGF enhanced E-cadherin reduced (from 50 to 80%) by the combined exposure to miR-125b plus NS-398, while the single treatment miR- in 1833 cells transfected with miR-CTR, and miR-125b 125b or NS-398 seemed less effective. reduced E-cadherin under basal and HGF conditions. Altogether, our findings suggested a key role of the Hypoxia induced MMP2 and vimentin, while being genes examined in bone metastasis growth, and the effi- prevented by concomitant miR-125b transfection. Also, cacy of the combined treatment tested. miR-125b showed inhibitory effects on MMP2 and vimentin under 0.1% FBS, and HGF caused a reversion Expression of ETS1, HIF1A and PTGS2 and their (Fig. 6a). intracellular distribution in bone metastasis under miR- TOPFLASH-gene reporter was used to evaluate WNT 125b plus NS-398 pathway activity (Fig. 6b). miR-125b alone inhibited of Immunohistochemistry images of the genes examined about 40% the luciferase activity, while in the presence of are shown in Figs. 3–5. As reported in Fig. 3 and in hypoxia or HGF a reversion of TOPFLASH activity was Supplementary Figure S2, the expression of ETS1 was observed, peaking (2.5-fold) under hypoxia. very high in bone-metastasis both at cytosol and nuclear In the nuclei of the cells transfected with miR-CTR, the levels (inset). ETS1 was elevated also in the cytosol of long-time exposure to hypoxia gave a huge HIF1A supportive cells of the bone marrow, while in the normal increase, and HGF tripled HIF1A: these increases were bone the ETS1 signal was scarce (Supplementary Fig- partly prevented by the concomitant miR-125b treatment. ure S2). The miR-125b plus NS-398 treatment reduced The transfection of miR-125b under 10% FBS (upper mostly metastatic ETS1, including the nuclear signal panel) and 0.1% FBS (lower panel) doubled HIF1A in the (Fig. 3, insets). As shown in Fig. 4, in bone metastases cytosol (Fig. 6c). under the combined treatment, the expression of HIF1A Altogether, the 1833 cells required the serum presence largely diminished at cytosol and nuclear levels (insets); for the miR-125b-dependent stimulation of the epithelial the signal of HIF1A extensively decreased also in the marker E-cadherin, while the miR-125b negative effects cytosol of the bone marrow supportive cells. PTGS2 signal towards the mesenchymal markers MMP2 and vimentin, in bone metastases was mild, showing prevalent cytosolic the staminal WNT pathway and nuclear HIF1A occurred localization, and it was low in the bone marrow: the both under 10 and 0.1% FBS. Also, the reduction of Official journal of the Cell Death Differentiation Association Maroni et al. Cell Death and Disease (2018) 9:472 Page 5 of 13 Fig. 3 miR-125b in combination with NS-398 affected ETS1 expression in bone metastasis of xenograft mice. We show representative images of ETS1 in bone metastasis from mice treated with the combination miR-125b plus NS-398, or not (ME). Serial sections for each specimen from three mice at 26 days were analyzed obtaining similar results. me, metastasis; bm, bone marrow; bo, bone. Scale bar = 120 μm (reported in 40× magnification panels). Scale bar = 60 μm (reported in the three insets). The negative control was performed without the specific antibody Official journal of the Cell Death Differentiation Association Maroni et al. Cell Death and Disease (2018) 9:472 Page 6 of 13 Fig. 4 HIF1A expression in bone metastasis of xenograft mice under miR-125b in combination with NS-398. We show representative images of HIF1A in bone metastasis of mice treated with the combination miR-125b plus NS-398, or not (ME). Serial sections for each specimen from three mice at 26 days were analyzed obtaining similar results. me, metastasis; bm, bone marrow; bo, bone. Scale bar = 120 μm (reported in an exemplificative panel). The negative control was performed without the specific antibody nuclear HIF1A by miR-125b in the presence of hypoxia or Figure 7a shows that 36-h hypoxia doubled the ETS1 HGF, was consistent with the immunohistochemistry protein level in 1833 cells, but not when miR-CTR was findings (Supplementary Figure S4). transfected. miR-125b in the presence or the absence of Official journal of the Cell Death Differentiation Association Maroni et al. Cell Death and Disease (2018) 9:472 Page 7 of 13 Fig. 5 miR-125b in combination with NS-398 affected PTGS2 expression in bone metastasis of xenograft mice. We show representative images of PTGS2 in bone metastasis of mice treated with the combination miR-125b plus NS-398, or not (ME). Serial sections for each specimen from three mice at 26 days were analyzed obtaining similar results. me, metastasis; bm, bone marrow; bo, bone. Scale bar = 120 μm (reported in an exemplificative panel). The negative control was performed without the specific antibody hypoxia down-regulated ETS1 (p51/52), while the p27 (Fig. 7b). The consensus sequences for transcription factors form of ETS1 was unchanged by the treatments tested. in osteocalcin and SPARC promoters are reported in Sup- PTGS2, SPARC and osteocalcin were induced by hypoxia plementary Figure S6: the numerous ETS1 and Runx2 in 1833 cells transfected or not with miR-CTR: miR-125b binding sites shown might explain the efficacy of the largely prevented the stimulatory effects of hypoxia treatments affecting metastasis phenotype. Putative binding Official journal of the Cell Death Differentiation Association Maroni et al. Cell Death and Disease (2018) 9:472 Page 8 of 13 Fig. 6 Effect of miR-125b on epithelial and mesenchymal markers and on HIF1A expression under hypoxia or HGF in 1833 cells. a Histograms of Western blots densitometric data; the experiments were repeated three times and the means ± S.E. are shown. Vinculin was used for Δ ΔΔ normalization. P < 0.05, P < 0.005 vs. miR-CTR; *P < 0.05 vs. miR-125b. Also, we report the representative images of the Western blots. b TOPFLASH luciferase activity is shown. The assays were performed in triplicate, and were repeated three times; the data are shown as the means ± S.E. P < 0.05 vs. miR-CTR; *P < 0.05 vs. miR-125b. c Representative images of Western blots performed in triplicate are shown. Vinculin and B23 were used for Δ ΔΔ normalization. The data of densitometric analysis are reported in the table as fold-variations vs. the first lane, considered 1. P < 0.05, P < 0.005 vs. § §§ miR-CTR; *P < 0.05 vs. miR-CTR plus hypoxia or plus HGF; P < 0.05, P < 0.005 vs. miR-125b sites of Twist and Snail were also present in the two gene of ETS1. In 1833 cells, HGF induced the dominant promoters, suggesting a relationship between the changes of negative p27, which might interfere in ETS1 activity gene expression and the metastatic phenotype. (Fig. 7c). miR-125b transfection, in the presence or the absence of The pattern of ETS1 under HGF or hypoxia in vitro was HGF, down-regulated both the p51/52 and the p27 forms in agreement with that observed in vivo, indicating the Official journal of the Cell Death Differentiation Association Maroni et al. Cell Death and Disease (2018) 9:472 Page 9 of 13 Fig. 7 A gene pattern for adhesion/invasion of 1833-bone metastatic cells was affected by miR-125b under hypoxia, or HGF treatment. a Representative images of Western blots repeated three times are shown. Vinculin was used for normalization. The data of densitometric analysis are Δ § reported in the table as fold-variations vs. the first lane, considered 1. *P < 0.05 vs. untreated 1833 cells; P < 0.05 vs. miR-CTR; P < 0.05 vs. miR-CTR under hypoxia. b Representative images of Western blots repeated three times are shown. Vinculin was used for normalization. The numbers at the Δ ΔΔ § bottom indicate the fold-variations vs. the first lane, considered 1. *P < 0.05, **P < 0.005 vs. untreated 1833 cells; P < 0.05, P < 0.005 vs. miR-CTR; P < 0.05 vs. miR-CTR under hypoxia. c Representative images of Western blots repeated three times are shown. Vinculin was used for normalization. The data of densitometric analysis are reported in the table as fold-variations vs. the first lane, considered 1. *P < 0.05 vs. untreated 1833 cells; P < 0.05 vs. miR-CTR; P < 0.05 vs. miR-CTR under HGF. d Representative images of Western blots repeated three times are shown. Vinculin was used for Δ ΔΔ normalization. The numbers at the bottom indicate the fold-variations vs. the first lane, considered 1. *P < 0.05 vs. untreated 1833 cells; P < 0.05, P < 0.005 vs. miR-CTR; P < 0.05 vs. miR-CTR under HGF. e To make a comparative evaluation of the gene effects of miR-125b under hypoxia or HGF, we show the histograms of the densitometric values of the Western blots for PTGS2, SPARC and osteocalcin. The data are the means ± S.E. of three Δ ΔΔ § separate experiments. P < 0.05, P < 0.005 vs. respective miR-CTR transfected cells; P < 0.05 vs. respective miR-125b transfected cells Official journal of the Cell Death Differentiation Association Maroni et al. Cell Death and Disease (2018) 9:472 Page 10 of 13 important role played by these microenvironmental sti- muli in the inhibitory effect of miR-125b towards ETS1 in the xenograft model (Supplementary Figure S3). Only under HGF but not in hypoxic conditions the ETS1-p27 form decreased when the 1833 cells were transfected with miR-CTR or miR-125b. PTGS2 and SPARC were induced by miR-125b plus HGF; osteocalcin was strongly down-regulated by star- vation, and incremented under miR-125b plus HGF (Fig. 7d). In conclusion, in the histograms (Fig. 7e) we compared the effects of miR-125b under the two microenviron- Fig. 8 Effects of miR-125b on gene expression under mental stimuli on PTGS2, SPARC and osteocalcin. microenvironmental stimuli in bone metastasis SPARC is a matricellular protein, not really an ECM component, which might participate in the stiffness and the release of growth factors , initiating changes in neoplastic cell phenotype with an involvement of miR- at early stages after bone engraftment of DTC. The NAs . Osteocalcin is important for the osteoblastic therapeutic approach consisting in the overexpression of mineralization of endosteal-like ECM: the endosteum miR-125b which targets a wide spectrum of genes, such as lines the inner wall of long bones surrounding the bone ETS1 and PTGS2, will eventually impact clinical practice marrow cavity, and drives the cellular changes for osteo- preventing bone resorption. The exit from quiescence/ 4,33 lytic metastases . Normally, osteocalcin is produced dormancy depends on osteoclast activation, and also on exclusively by osteoblasts, acting in the bone matrix as a stroma composition and neoangiogenic vessels . non-collagenous protein, and is now considered an inhi- The molecular mechanisms underlying the therapeutic bitor of bone mineralization even if opposite hypotheses efficacy of the studied treatment miR-125b plus NS-398 cannot be excluded . By considering PTGS2 and SPARC, would be multiple (Fig. 8). First, the blockade of the miR-125b was stimulatory in the presence of HGF and autoregulatory loop between ETS1 and HIF1A/HIF1, inhibitory under hypoxia. Osteocalcin seemed strongly because of the reciprocal control of these transcription hampered by miR-125b, mostly under starvation, with a factor activities , and second a complete inhibition of partial reversion both under HGF and hypoxia. Thus, the PTGS2 by the concomitant chemical and miR-125b effect of miR-125b on target genes seemed to depend on treatment. At support, PTGS2 signal remarkably microenvironmental stimuli. decreased in osseous metastases and the bone marrow, also due to the down-regulation of HIF1A throughout the Discussion metastatic tissue: the alpha/beta heterodimer HIF1 is a In the 1833 xenograft model of skeleton metastasis from transcription factor known to target PTGS2 and 20,25 breast carcinoma the concomitant exposure to miR-125b ETS1 . Our proposed therapy would have a direct mimic and NS-398, a specific PTGS2 chemical inhibitor, effect on PTGS2, through miR-125b action on HuR sites, reduced and delayed the outgrowth of osteolytic bone even if a post-transcriptional effect of miR-125b on ETS1 metastases prolonging mice survival in respect to the cannot be excluded. Also, an indirect effect of the therapy single treatments. The miR-125b strongly enhanced the on ETS1 through HIF1A downregulation might occur, life-protective effect of NS-398, and we hypothesize that consistent with the decreases of ETS1 and HIF1A in the efficacy of the combined treatment depended on the cytosol and nuclei of metastases and in the bone marrow. reduction of chemoresistance, known to be associated We suggest that these amplifying effects might impinge 7,18 with EMT . The endpoint of our study was to on invasion and angiogenesis, because ETS1 and PTGS2 demonstrate that miR-125b enhances the epithelial-like are key players in these processes: PTGS2 through pros- transition in vitro, and that the paracrine stimuli and taglandin E2 regulates HIF1A nuclear translocation and 20,25 physical conditions might counteract or favor the phe- HIF1 activity (Supplementary Figure S4) . Capillaries notypic changes, explaining also the in vivo findings on are associated with quiescent and actively resorbing growth and gene expression in bone metastases. endosteal surfaces, and are implicated in development of Importantly, the adverse effect of miR-125b mimic plus bone metastasis, permitting the access of DTC to bone- NS-398 on metastatic growth started at 4 days from 1833/ remodeling compartment or to the immediate adjacent TGL-cell injection and became highly significant at microenvironment . 9 days, leading to suppose that the hampering of coloni- Third, in vivo phenotypic changes under miR-125b zation was caused by molecular and cellular modifications plus NS-398 seemed to depend on the metastatic Official journal of the Cell Death Differentiation Association Maroni et al. Cell Death and Disease (2018) 9:472 Page 11 of 13 down-regulations of ETS1 and HIF1A, which are critical the stimulatory effect of hypoxia. Therefore, the miR-125b 20,39 for the invasive phenotype and staminality . Also, in mimic might reduce bone metastasis colonization in vivo agreement with the hypothesis that in 1833-xenograft influencing SPARC roles in metastasis plasticity and in model miR-125b decreased the metastasis resistance to metastasis dormancy, by the regulation of biological and 34,36,46,47 chemotherapy, the microenvironmental stimuli would be physical properties of the ECM . HIF1A beyond critical for the effect of miR-125b in vivo, suggesting that SPARC participates in metastasis dormancy . the bone context encountered by DTC would be funda- In conclusion, a strong down-regulation of osteocalcin mental for their fate. The skeleton microenvironment is was observed in the presence of miR-125b under serum modified by the engraftment of metastatic cells influen- deprivation, indicating the susceptibility to growth factors, 18,37 cing the EMT , and it would interfere in the therapies while HGF restoration not only reversed osteocalcin such as that with miR-125b mimic. expression, but also increased PTGS2 and SPARC under The axis HGF/Met receptor seems to be implicated in miR-125b. The miR-125b-dependent impairment of 20 38 stroma-induced drug desensitization , and hypoxia con- osteocalcin, considered an inhibitor of mineralization , ferring stem like properties might give resistance to might be related to protection from osteolysis: this effect therapy . Consistently, we found that the tumor sup- would be partly prevented by the microenvironmental pressor gene miR-125b in 1833 cells reduced WNT stimuli. The combined therapeutic approach miR-125b activity and targeted transcription factors, such as ETS1, plus NS-398 seems promising for the early hampering HIF1 and WNT, likely affecting gene expression in bone effects on metastatic growth and osteolytic lesions, con- metastasis. Numerous ETS1 consensus sequences are sidering also that the initial metastatic steps and therapy present in SPARC and osteocalcin promoters (Supple- efficacy are strongly influenced by the microenvironment mentary Figure S6). In addition, considering that in bone- conditions. metastatic cells miR-125b enhanced the expression of E- cadherin, while decreasing MMP2 and vimentin, our Materials and Methods present results suggested a switch towards an epithelial Cell transfection phenotype, which is known to be more responsive to The 1833 bone metastatic clone, derived from MDA- chemotherapy than the mesenchymal/invasive MB-231 breast adenocarcinoma cells, and the 1833 cells 7,18,20 phenotype . retrovirally transfected with HSV1-tk/GFP/firefly lucifer- In bone metastatic cells, which show an epithelial phe- ase construct (1833/TGL) , were a gift from Dr. J. Mas- notype , miR-125b might antagonize the function of sagué (Memorial Sloan-Kettering Cancer Center, New miR-200 : we propose that miR-125b by blocking p53 York, NY, USA). 11,19,37 prevents miR-200-inhibitory function . As a con- The cells, routinely maintained in DMEM containing sequence, Twist activity might mediate the MET meta- 10% FBS (Sigma-Aldrich, Saint Louis, MO), were used stable phenotype with E-cadherin expression . The miR- after two or three passages in culture. The 1833 and 1833/ 125b effect on the gene expression of bone metastatic TGL cells were infected with miR-125b expression lenti- cells depended on serum conditions, HGF and hypoxia virus (hsa-miR-125b-5p MIMAT0000423, GenTarget exposure, favoring the reduction of ETS1 expression Inc., San Diego, CA, USA) or with miR-CTR (negative in vitro, while the microenvironmental stimuli counter- control, empty miR lentivirus, GenTarget Inc.), following acted the miR-125b-dependent epithelial phenotype with the manufacturer’s recommended procedures. Lentiviral E-cadherin reduction by hypoxia and MMP2 and vectors co-expressed puromycin resistance for the selec- vimentin enhancements by HGF. tion of infected cells. A pilot experiment has been done to Altogether, the knowledge of phenotypic changes under determine the antibiotic’s kill curve for our cells. Thus, therapy were further deepened by studying SPARC and 72-h infected cells were selected with 2 μg/ml puromycin, osteocalcin, which are target genes of Runx2 transcription and were maintained under selection in all the 34,41 factor ; HIF1A expression is also controlled by Runx2 experiments. by preventing the ubiquitination . Runx2 plays key roles In another group of experiments, flasks containing 1833 43,44 in bone metastasis from breast carcinoma , being cells at 70% of confluence were used to transfect the 35,45 regulated by Endothelin-1, HGF and hypoxia . Nota- TOPFLASH gene reporter from Dr. B.M. Gumbiner bly, bone-metastatic cells acquire osteomimicry by pro- (Memorial Sloan-Kettering Cancer Center, New York, ducing SPARC and osteocalcin, as shown in the 1833- NY, USA). For the transfection of the TOPFLASH con- 32,34,46 xenograft model . struct, we performed an incubation mixture 3:1, DNA: HGF and hypoxia caused SPARC accumulation, which Fugene 6 (Roche Diagnostics, Monza, Italy); the mixture might be important to determine the adhesive properties contained also the internal control pRL-TK (Renilla of bone metastasis in the osteoblastic niche, ECM stiffness Luciferase Plasmid). Firefly/Renilla luciferase activity and growth-factor release, and miR-125b counteracted ratios were calculated by the software . Official journal of the Cell Death Differentiation Association Maroni et al. Cell Death and Disease (2018) 9:472 Page 12 of 13 Animal treatment Transduction Laboratories, Bedford, MA, USA), anti- Animal studies were carried out according to the MMP2 (1:500, Novus Biologicals, Abingdon, UK), anti- Institutional Guide for Care and Use of Laboratory vimentin (1:500 Santa-Cruz Biotechnology), anti-SPARC Animals, and the Directive 2010/63/UE. All mice were (1:200, H-90 Santa Cruz Biotechnology), anti-osteocalcin bred and maintained under specific pathogen-free con- (5 μg/ml, Abcam, Cambridge, UK), anti-B23 (1:1000, ditions in the institutional animal facility of the Scientific H106 Santa Cruz Biotechnology), or anti-vinculin (1:1000, Cell Signaling, Leiden, The Netherlands) was used for Institute San Raffaele, Milano, Italy. All experiments were performed with the protocol approved by the Institutional immunoblotting. Densitometric analysis was performed Animal Care and Use Committee of Scientific Institute after reaction with ECL plus chemiluminescence kit from San Raffaele and by Ministero della Salute. Anaesthetized Thermo-Fisher Scientific (Waltham, MA, USA) or Clarity nu/nu mice were treated as follows: one animal group Max Western ECL Substrate-Luminol Solution (Biorad, (ME, n = 11) was injected with the 1833/TGL cells; a Hercules, CA, USA). second group (ME + miR-125b, n = 11) received 1833/ TGL cells infected with miR-125b; a third group (ME + Statistical analysis miR-125b + NS-398, n = 10) received 1833/TGL cells The statistical analysis of the values of bioluminescence infected with miR-125b, and the mice were concomitantly for the xenograft mice groups, as well as of Western injected with 20 mg/kg NS-398 i.p. 5 days/week until the blots densitometry and luciferase activity values was suppression; the fourth group was treated only with performed by analysis of variance. The number of NS-398 (n = 10); the fifth group was administered independent experiments has been indicated in the 1833/TGL cells infected miR-neg-CTR (ME + miR-CTR, Legends of the Figures. The data are shown as means ± n = 4). For the xenograft model preparation, 5 × 10 S.E., and their significance was evaluated on the infected or non-infected cells were injected to the mice, original values in the case of fold-variations. P < 0.05 was that were monitored by Optical imaging and μCT assay , considered significant. For xenograft mice, the survival and were sacrificed to prevent the suffering. Three ani- data were analyzed by Kaplan–Meier method and the mals per group were sacrificed at the indicated times to Log-rank (Mantel–Cox) test. P < 0.05 was considered perform immunohistochemistry and Hematoxylin & significant. Eosin assays. Acknowledgements The injection efficiency for all the mice was controlled This work was supported by grants from Ministero della Salute Ricerca by monitoring the bioluminescence signal 1 h after Corrente L4077, L4084 and L4101. xenografting. We normalized the data of bioluminescence Author details for each animal with the value obtained at 24 h. This Istituto Ortopedico Galeazzi IRCCS, Via R. Galeazzi 4, 20161 Milano, Italy. procedure was made to exclude a potential impact of the 2 Dipartimento di Scienze Biomediche per la Salute, Molecular Pathology pretreatments, i.e., miR-125b-cell transfection or NS-398 Laboratory, Università degli Studi di Milano, Via L. Mangiagalli 31, 20133 Milano, Italy exposure, on extravasation and homing with an inter- ference in the evaluation of metastasis growth. Conflict of Interest The authors declare that they have no conflict of interest. Immunohistochemistry Bone samples fixed with 10% formalin, were decalcified, Publisher's note embedded in paraffin, and serial sections were prepared . 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